The Pyruvate Carboxylase (PC) Project
Updated Jan. 2018
Pyruvate carboxylase (PC) catalyzes the biotin-dependent carboxylation
of pyruvate to produce oxaloacetate. PC has crucial roles in
gluconeogenesis, lipogenesis, glyceroneogenesis, and insulin secretion in mammals.
PC deficiencies are linked to lactic acidemia, hypoglycemia, and other diseases.
Four single-site mutations, V145A, R451C, A610T, M743I, are associated with
PC is a multi-domain enzyme of about 130 kD in eukaryotes and most bacteria.
It contains the biotin carboxylase (BC), carboxyltransferase (CT), and biotin-carboxyl
carrier protein (BCCP) domains.
The BC domain
catalyzes the first step of the reaction: the carboxylation of the
biotin prosthetic group that is covalently linked to BCCP.
In the second step of the
reaction, the carboxyltransferase (CT) domain catalyzes the
transfer of the carboxyl group from (carboxy)biotin to pyruvate.
PC is only active in the tetrameric form. The catalytic activity is stimulated
by acetyl-CoA and inhibited by aspartate.
Major findings from this project
- The crystal structure of the CT+PT+BCCP domain of human PC has been
determined at 2.8A resolution.
- The crystal structure of full-length S. aureus PC has been
determined at 2.8A resolution.
- The structures identify a novel domain, the PC tetramerization (PT) domain,
that is important for the tetramerization of this enzyme.
- Mutation of a single residue in the PT domain, F1077A, can disrupt the tetramer
and inactivate the enzyme.
- The BCCP domain is swapped between dimers in the tetramer, explaining why
only tetramers of the enzyme are active.
- The active sites of the BC and CT domains are separated by about 75 A, suggesting
that the entire BCCP domain will need to translocate during the reaction.
- BCCP-biotin is observed in the active site of the CT domain, providing the first
molecular insight into how biotin participates in the CT reaction.
- The A610T mutation is likely to block biotin binding in the CT active site, while
the M743I mutation is likely to block pyruvate binding.
- The R451C mutation is located in the acetyl-CoA binding site, and de-sensitizes the enzyme
towards this activator.
- The V145A mutation is in the BC domain, and is known to destabilize the enzyme.
- The Thr908 residue is likely to play a catalytic role in the CT reaction.
- There are dramatic conformational differences to the structure of R. etli PC, obtained in
complex with the activator ethyl-CoA. The structural differences may be due to the binding of
- An exo binding site for biotin is observed, located at the PT-CT domain interface. The
functional relevance of this site is currently not known.
- The structure of S. aureus PC in complex with CoA is symmetrical, with 4 CoA binding site in the tetramer.
L. monocytogenes PC (LmPC) is inhibited allosterically by the bacterial second messenger
cyclic-di-AMP (c-di-AMP, cdA).
The structure of LmPC in complex with c-di-AMP has been determined at 2.5 A resolution. The
structure of LmPC alone has been determined at 3.3 A resolution.
c-di-AMP is bound at the dimer interface of the CT domain of LmPC, about 25 A away from the active
site, confirming its allosteric mode of action.
The stoiochiometry of the complex is 4:2, 4 LmPC monomers, and 2 c-di-AMP molecules.
Residues in the binding site show variations in HsPC, SaPC and other enzymes, consistent with their being insensitive to c-di-AMP. On the other hand, the PC from the human pathogen E. faecalis
has most of the residues conserved, and is sensitive to c-di-AMP.
There are large conformatinal differences between the free LmPC structure and the c-di-AMP complex.
The binding site for the compound does not exist in the free enzyme.
The conformations of the four LmPC monomers in the c-di-AMP complex are essentially identical.
In contrast, the conformations of the four monomers in the free enzyme show large differences.
It may be possible that the PC enzyme needs to go through conformational rearrangements during
catalysis, and c-di-AMP inhibits the enzyme by 'freezing' it in a single state.
The regulation of LmPC by c-di-AMP is important for maintaining metabolic balance, and
for intracellular growth and infectivity by the bacterium.
Publications from this project
S. Xiang & L. Tong. (2008).
Crystal structures of human and Staphylococcus aureus pyruvate carboxylase
and molecular insights into the carboxyltransfer reaction.
Nature Struct. Mol. Biol., 15, 295-302.
L.P.C. Yu, S. Xiang, G. Lasso, D. Gil, M. Valle & L. Tong. (2009).
A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A.
Structure, 17, 823-832.
G. Lasso, L.P.C. Yu, D. Gil, S. Xiang, L. Tong & M. Valle. (2010).
Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase.
Structure, 18, 1300-1310.
G. Lasso, L.P.C. Yu, D. Gil, M. Lazaro, L. Tong & M. Valle. (2014).
Functional conformations for pyruvate carboxylase during catalysis
explored by cryoelectron microscopy.
Structure, 22, 911-922.
K. Sureka,* P.H. Choi,* M. Precit, M. Delince, D.A. Pensinger,
T.N. Huynh, A.R. Jurado, Y.A. Goo, M. Sadilek, A.T. Iavarone,
J.-D. Sauer, L. Tong$ & J.J. Woodward.$ (2014).
The cyclic dinucleotide c-di-AMP is an allosteric regulator of
metabolic enzyme function.
Cell, 158, 1389-1401. (*-equal first authors, $-co-corresponding authors)
P.H. Choi, J. Jo, Y.C. Lin, M.-H. Lin, C.-Y. Chou, L.E.P. Dietrich
& L. Tong. (2016).
A distinct holoenzyme organization for two-subunit pyruvate carboxylase.
Nature Commun. 7, 12713.
A.T. Whiteley, N.E. Garelis, B.N. Peterson, P.H. Choi, L. Tong,
J.J. Woodward & D.A. Portnoy. (2017).
c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth,
antibiotic resistance, and osmoregulation.
Mol. Microbiol., 104, 212-233.
P.H. Choi, T.M.N. Vu, H.T. Pham, J.J. Woodward, M.S. Turner
& L. Tong. (2017).
Structural and functional studies of pyruvate carboxylase regulation
by cyclic-di-AMP in lactic acid bacteria.
Proc. Natl. Acad. Sci. USA, 114, E7226-E7235.
Funding for this project
NIH R01DK067238 (2004-2013)
NIH R01AI116669 (PI: Woodward, 2015-)
© copyright 2008-2018, Liang Tong.